PROJECT SUMMARY Multidrug resistant-gonorrhea is a serious global health threat. Penicillin Binding Protein-targeting ?-lactams have long been the front line therapeutic option for gonorrhea, but are now in serious jeopardy. Resistance to the last remaining front line ?-lactams, the extended spectrum cephalosporins, has steadily increased over the past decades resulting in the CDC eliminating cefixime as a therapeutic option in 2012. Now only ceftriaxone remains, but a series of resistant clinical isolates exhibiting PBP2-target based resistance have emerged in the past decade, forecasting its imminent failure. As few clinical development candidates addressing MDR- gonorrhea are in the drug development pipeline, and vaccine development is unlikely to be a solution due to high antigenic variability in clinical isolates, there is an urgent need for new therapeutic options. A new chemical series maintaining PBP target inhibition represents a promising strategy, enabling new combination therapies to minimize further evolution of resistance. VenatoRx has identified a novel chemical series of reversible covalent non-?-lactam Penicillin Binding Protein inhibitors impervious to the action of ?-lactamases that are being optimized to address altered PBP2 targets responsible for ESC-resistance in Neisseria gonorrhoeae. Significant strides in microbiological activity have been achieved with the lead compound VNRX-6355 in MDR-gonorrhea isolates including a mosaic PBP2-producing H041 clinical isolate. Improved binding to altered PBP2 variants by VNRX-6355 has provided a >128-fold improvement in microbiological activity in H041 over initial hit compounds in the series (shift in MIC from >512 mg/L to 4 mg/L) and is only 8-fold away from our MIC target to obtain an MIC90 of ?0.5 mg/L. These compounds are highly selective, rapidly bactericidal and efficacious in a murine septicemia model of E. coli infection. The goal of this proposal is to select a potent preclinical candidate addressing PBP2 target variants in ESC-resistant gonorrhea, confirm favorable PK properties for intramuscular administration, select the first preclinical development candidate, perform preclinical IND-enabling studies and file an IND with the US FDA. Ultimately this approach is intended to be a long term strategy to safeguard PBP- targeting in gonorrhea treatment by preventing the expansion of ?-lactamases in Neisseria gonorrhoeae that would inevitably evolve from more effective targeting of PBP2 variants by current or newly optimized ?-lactam- based strategies.